This Competitive Revision Application is being submitted in response to Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications, with respect to the Parent Grant R01DE16132, "Dental stem cells and tooth tissue engineering". The ultimate goal of this research project is to develop novel biologically based, dental and craniofacial skeletal repair and regeneration therapies in humans. We hypothesize that improved knowledge of dental stem cell (DSC) properties, and characteristics, combined with improved knowledge of tissue engineering strategies to reliably generate mineralized dental and craniofacial tissues of predictable size and shape, will result in the development of novel and effective, clinically relevant therapies for humans. To address this hypothesis, in the studies proposed here we will characterize 1) naturally formed third molar wisdom tooth extracellular matrix (ECM) components and organization, and 2) also examine the use of two novel hydrogel scaffolds, using both in vitro and in vivo approaches, to expand the original aims of the Parent Grant. These studies will be performed in collaboration with two new collaborators, Drs. Ali Khademhosseini, Harvard Medical School, and Glenn Prestwich, University of Utah. Our novel approach to tooth repair and regeneration, using human DSCs, and state of the art scaffold fabrication methods, combined with our extensive expertise in Developmental Biology and Tissue Engineering, have the potential to provide new and improved, biologically based repair and regeneration strategies, using autologous tissues. The successful accomplishment of the proposed studies would dramatically alter the field of dentistry as it currently exists, extending clinically relevant dental repair therapies to include biologically based dental materials with properties closely matching those of naturally formed dental tissues. PUBLIC HEALTH RELEVANCE: Project Narrative. Due to the severely limited ability for craniofacial and dental tissues to repair themselves, these defects remain a significant clinical problem that affects millions of people worldwide, including both children and adults. Tooth decay, periodontal disease, craniofacial birth defects, trauma, and cancer, all contribute to compromised oral health, and correspondingly to reduced overall health and quality of life. At the present time, synthetic material based repair methods, or autologous grafts which often result in donor site morbidity, are exclusively being used to repair human craniofacial skeletal and tooth defects. The studies proposed here will expand on our prior published studies, to devise methods to repair and regenerate craniofacial skeletal and tooth defects using biologically based tissue engineering approaches, and human dental stem cells. We anticipate that the successful development of alternative biologically based tissue repair methods will provide significantly improved therapies for individuals requiring repair/regeneration of craniofacial skeletal and tooth defects.